Treating compositions with lactoferrin

ABSTRACT

Reducing the microbial contamination of a composition by treating with lactoferrin.

RELATED APPLICATIONS

[0001] This application is a division of application Ser. No.09/932,190, filed Sep. 17, 2001, which is a division of application Ser.No. 09/421,632, filed Oct. 19, 1999 (now U.S. Pat. No. 6,277,817), whichis a division of application Ser. No. 08/724,586, filed Aug. 30, 1996(now U.S. Pat. No. 6,066,469), which is a continuation of applicationSer. No. 08/238,445, filed May 5, 1994 (now abandoned), which is acontinuation-in-part of application Ser. No. 08/132,218, filed Oct. 6,1993 (now Abandoned), which is a continuation of application Ser. No.07/998,645, filed Dec. 30, 1992 (now abandoned), which is a continuationof application Ser. No. 07/489,186, filed Mar. 8, 1990 (now abandoned),the disclosure of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the glycoproteinlactoferrin, its production, purification, and uses. BACKGROUND OF THEINVENTION

[0003] Lactoferrin plays an important role in iron transport andutilization in humans. A glycoprotein containing about 6% sugar andhaving a total molecular weight of about 80,000 daltons, humanlactoferrin is capable of binding two ferric ions with high affinity.Although lactoferrin binds iron tightly, the binding is reversible sothat the metal is available upon demand to cells with a need for thisessential element. The metal is captured by the side chains of specificamino acids: two tyrosines, one histidine and one aspartate which incombination form a cleft in the surface of the protein. That portion ofthe protein which contains the aforesaid four amino acids and forms thecleft is termed the “iron-binding domain.” Each natural lactoferrinmolecule has two iron-binding domains.

[0004] Human milk is high in lactoferrin content. The high degree ofiron absorption from human milk is manifested in a low incidence of irondeficiency anemia among breast fed infants compared to infants fed withcow's milk. Accordingly, lactoferrin is a key protein for healthydevelopment of infants. Lactoferrin also plays an important role incell-mediated host immunity. It is present in high concentrations in allbodily secretions, such as tears, sweat, and ciliated respiratorymucous. Because it sequesters iron, lactoferrin can neutralizepathogenic microorganisms by preventing them from obtaining necessaryiron at the site of entry, thereby preventing the spread of infection.

[0005] Although iron is an essential material in humans, excess iron inthe body can induce pathological conditions as well. Chronic ironoverload, known as hemosiderosis, is characterized by a greater thannormal local or generalized deposition of iron within certain bodytissues. Lactoferrin helps to manage the balance of free iron in thebody to prevent occurrence of such pathological states in healthyindividuals.

[0006] The severely limited amount of human milk, the major source ofhuman lactoferrin, restricts lactoferrin production. Furthermore,production of lactoferrin from human milk presents a risk factor ofinfectious contamination. That is, it could carry with it a potentiallylethal contaminant, such as the human immunodeficiency virus (HIV) oranother undesirable agent.

SUMMARY OF THE INVENTION

[0007] Accordingly, the present invention provides for the cloning andexpression of human lactoferrin using recombinant DNA techniques. Thelactoferrin of the present invention is free of naturally occurringcontaminants, e.g., proteins and viruses, that would be detrimental tothe recipient.

[0008] In one embodiment of the present invention there is provided agene comprising a DNA molecule encoding human lactoferrin protein. Moreparticularly, the DNA molecule comprises the nucleotide sequence (Seq.ID No. 1) and the protein comprises the amino acid sequence (Seq. ID No.2) as substantially depicted in FIG. 3. An expression system is providedfor expressing the gene encoding the protein. Preferably, the expressionsystem is a plasmid. Also described herein, is a host cell linetransformed with the gene of the present invention, i.e., the geneencoding human lactoferrin. Preferably the host cell organism is aeukaryotic cell.

[0009] In another embodiment of the invention, there is provided amethod of producing human lactoferrin comprising the steps of (a)isolating a gene encoding human lactoferrin; (b) transforming a hostcell with the gene; (c) culturing the transformed cells which expressthe gene product; and (d) collecting lactoferrin from the cells.

[0010] In a further embodiment of the invention there is described achromatography method for purifying lactoferrin protein, and otherproteins, comprising the steps of (a) contacting a substance with afirst adsorbent to obtain adsorbed and non-absorbed fractions; (b)eluting the adsorbed fraction with an eluant; and (c) contacting theadsorbed fraction with a second adsorbent, wherein the improvementcomprises equilibrating the second adsorbent with the eluant followed bycontacting the eluate containing the adsorbed fraction with the secondadsorbent.

[0011] In still a further embodiment of the invention there is provideda method for inhibiting microbial growth in a mammal comprisingtopically or systemically applying to a subject a therapeuticallyeffective amount of lactoferrin having less than about 25% metalloading; a method for inhibiting iron deficiency in a mammal comprisingorally administering a therapeutically effective amount of lactoferrinhaving at least about 35% iron loading; a nutritional supplementcomprising an iron-loaded human lactoferrin having at least about 35%metal loading and a nutritionally acceptable carrier or adjuvant; atopical antiseptic comprising a therapeutically effective amount oflactoferrin having less than about 25% metal loading and apharmaceutically acceptable carrier or diluent; and a method forinhibiting food spoilage comprising adding to the food an effectiveamount of lactoferrin having less than about 25% metal loading.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a schematic diagram showing the chromatography method ofthe invention.

[0013]FIG. 2 is a flow chart showing the purification of lactoferrin.

[0014]FIGS. 3a-3 d show the nucleotide sequence (Seq. ID No. 1) and thededuced amino acid sequence (Seq. ID No. 2) of the gene encoding humanlactoferrin.

[0015]FIG. 4 is an agarose gel analysis of amplified cDNA coding humanlactoferrin.

[0016]FIG. 5 is a map of the pUC118 plasmid.

[0017]FIG. 6 is a map of the pHIL-D1 plasmid.

[0018]FIG. 7 is a map of the pPIC9 plasmid.

[0019]FIG. 8 is a restriction fragment map of the cDNA lactoferrin gene.

[0020]FIG. 9 is an SDS-PAGE analysis of recombinantly expressedlactoferrin according to the present invention.

[0021]FIG. 10 is a Western blot analysis of the recombinantly expressedlactoferrin according to the present invention.

BREIF DESCRIPTION OF THE INVENTION

[0022] Lactoferrin is produced in accordance with the present inventionusing recombinant DNA technology to produce genetically modified DNAthat expresses lactoferrin. The recombinant DNA technology describedherein is standard technology in the art, such as described by Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989,Chapter 14, which disclosure is hereby incorporated by reference. Thatis, a polypeptide containing the primary structural conformation of thenaturally occurring human lactoferrin protein, having similar biological(i.e. physical) properties is produced. The DNA encoding lactoferrin ispreferably from a cDNA library derived from human RNA and ligated to anappropriate expression vector according to standard techniques in theart, such as those disclosed in Davis, et al., Basic Methods inMolecular Biology, Elsevier Science Publishing Co. (1986), whichdisclosure is hereby incorporated by reference. Preferably, the RNA isisolated from the human mammary gland and the preferred vector is phageλgt11. The cDNA library is screened for positive (DNA carrying thelactoferrin gene) clones using conventional techniques in the art, suchas disclosed by Davis, et al., supra, and Rado, et al., Blood, 70:4,989-993 (October, 1987), which disclosure is hereby incorporated byreference. For example, the cDNA is hybridized to a radiolabeledoligonucleotide probe and the positive clones identified by autoradiography. Preferably, positive clones are identified usinglactoferrin antisera, and an appropriate development system, such as anavidin/biotin reaction system. Large numbers of positive clones are thengenerated by infecting an appropriate bacterial host, such as E. coli Y1090, using methods that will be readily apparent to the skilled artisansuch as described by Davis, et al. (1986), supra. DNA is then isolatedfrom the clones. The cDNA encoding lactoferrin is thereafter cut with anappropriate restriction endonuclease such as EcoRI. The cut DNA encodinglactoferrin is separated by chromatography. Preferably, the separatedcDNA is further sub-cloned into another vector, such as the pGEM-4plasmid, and the inserted cDNA again excised and separated.

[0023] Expression of human lactoferrin according to the presentinvention is performed using an appropriate host organism, such as theyeast Pichia pastoris with an appropriate expression vector such asthose driven by the alcohol oxidase promoter and disclosed in Tschopp,Nucleic Acid Research, Vol. 15, pp. 3859-3876, (1987). Other usefulhosts include: (i) the yeast Hansenula polymorpha with an appropriateexpression vector driven by strong promoters such as methanol oxidase(MOX), dihydroxyacetone synthetase (DAS), or formate dehydrogenase(FMDH) as disclosed in Gellisen, Biotech. Adv., Vol 10, pp. 179-189,(1992) the disclosure of which is incorporated herewith by reference;and (ii) the Spodoptera frugiperda insect cells with an appropriateexpression vector such as a Baculovirus expression vector system drivenby polyhedrin promoter as disclosed in Gruenwald, Baculovirus ExpressionVector System: Procedures & Methods Manual, Second Edition, 1993 byPharmingen, the disclosure of which is incorporated by reference herein.

[0024] Insertion of the cDNA and expression of the human lactoferrin arecarried out using standard recombinant techniques that are readilyapparent to one skilled in the art, such as described in Rothstein,Methods in Enzymology, 101, 202-210 (1983); and Tschopp, et al.,Bio/Technology, 5, 1305-1308 (1987), which disclosures are herebyincorporated by reference.

[0025] In a preferred embodiment, to obtain the full length cDNA cloneof lactoferrin, the gene is amplified from a human mammary gland libraryusing the polymerase chain reaction (PCR) technique. Preferably,amplification of the insert encoding the lactoferrin gene is achieved byusing two synthetic oligonucleotide probes corresponding to the aminoacid sequence residues 1 to 9 of the amino terminus and residues 2070 to2079 of the carboxy terminus. The amplified insert is cut out withrestriction enzymes BamH I and Xba I, then purified, and ligated into anappropriate vector, preferably pBlueScripKS+, which is digested with thesame restriction enzymes. The lactoferrin gene is further subcloned intopUC118 vector, cut with Hind III and Sst I to produce a plasmiddesignated pUC118-LF, containing the coding region for the maturelactoferrin protein. Secretion of the lactoferrin protein may be furtherenhanced by further manipulations of plasmid pUC118-LF. For example, thefollowing two methods are provided for purposes of illustration.

[0026] The first method is the addition of a signal sequence to the 5′end of the clone. The preferred signal sequence is that described byPowell, et al., Nucleic Acids Research, 18, 4013 (1990), whichdisclosure is hereby incorporated by reference. pUC118-LF modified withthe aforementioned signal sequence is defined as pUC118-LFS. Preferably,the lactofernin gene, together with the signal sequence is cut frompUC118-LFS and then ligated to pHIL-D1 to produce pHIL-D1-LFS.

[0027] The second, and more preferable, method employs the alpha matingfactor pre-pro secretion signal. The mature lactofernin gene is cut fromplasmid pUC118-LF and ligated to plasmid pPIC9 which had been previouslyligated with an alpha mating factor pre-pro secretion signal.

[0028] An additional embodiment of the present invention relates tofragments of both the lactoferrin gene and lactoferrin. The active sitesof the lactoferrin protein are the iron-binding domains. Proteinsequences which contain one or more of the iron-binding domains of thelactoferrin protein sequester iron and, therefore, are useful in theantiseptic, dietary supplement, and food-spoilage retardant embodimentsof the present invention. The present invention contemplates a (DNA)fragment of the DNA molecule encoding human lactoferrin. The fragmentencodes a portion of the human lactoferrin protein containing at leastone of the iron-binding domains.

[0029] For example, a DNA fragment of the lactoferrin gene encoding onlyone iron-binding domain of human lactoferrin can be obtained through aspecific design of oligonucleotides for PCR amplification or through anantibody probe screening of a cDNA library, which procedures are readilyapparent to one skilled in the art. These fragments are also useful,e.g., as intermediates in the synthesis of the full-length gene andprotein.

[0030] Expression of human lactoferrin using the aforementioned modifiedplasmids is carried out according to techniques that will be readilyapparent to the skilled artisan, such as those disclosed in Rothstein(1983) and Tschopp, et al., (1987), supra.

[0031] Purification of the expressed protein according to the presentinvention is preferably carried out by one of several methods. In onepreferred embodiment, a cell-free culture media containing the expressedlactoferrin protein is passed through a filter that retains materialhaving a molecular weight greater than about 10,000 daltons and thensterilizing the retained protein. The material retained by the filter issubjected to a two-step affinity chromatography process. In the firststep, the affinity ligand is the reactive dye Cibacron blue F3G-A (colorindex (C.I.) 61211, λmax 605(374)mn) as disclosed by Bezwoda, et al.,Clin. Chim. Acta., 157, 89-94 (1986); and Chemical Abstracts Service(CAS) No. 12236-82-7, which disclosures are hereby incorporated byreference. Cibacron blue F3G-A can be covalently bound to a cross-linkedagarose gel by the triazine coupling method as described by Bohme, etal., J. Chromatography, 69, 209-214 (1972), which disclosure is herebyincorporated by reference. In the second step, controlled-pore glass(CPG) or silicic acid is used to further purify the adsorbed materialobtained in the first step.

[0032] In another preferred embodiment, the adsorbed material from theCPG or silicic acid is further chromatographed in a third step using oneof the following chromatography techniques before final filtration andsterilization: T-Gel chromatography; immobilized metal-ion affinitychromatography (IMAC) using a metal ion capable of forming a complexwith lactoferrin, such as nickel or copper, which can be coupled with animminodiacetic acid-epoxy activated gel (IDA Me(II)) as described bySulkowski, Frontiers in Bioprocessing, Sidkar et al., Ed., 343-353(1990), which disclosure is hereby incorporated by reference; orchromatography with the ligand phenyl glycidyl ether, which can becoupled to a cross-linked agarose gel as described by Janson, et al.,Protein Purification Principles High Resolution Methods andApplications, VSH Publishers, New York (1989), which disclosure ishereby incorporated by reference. The two-step and three-step methodspreviously described are schematically shown by the diagrams in FIG. 2.

[0033] The improved chromatography process of the present invention isuseful in purifying proteins, such as lactoferrin produced in accordancewith the present invention. As shown in FIG. 1, crude fermentation brothcontained in tank 1 passes to permeable membrane 3, which retainsmaterial having a molecular weight greater than 10,000 daltons andpasses an ultra filtrate containing water, salts, and lowmolecular-weight proteins. The retained material is washed with a bufferand further concentrated. The washed material is then applied tochromatography column 5 containing an adsorbent that has beenequilibrated with the buffer used to wash the filtered material whilevalve 7 is open and valve 9 is closed. After non-adsorbed material isdischarged through valve 7, valve 7 is closed and valve 9 opened.Adsorbed material is then eluted, and the eluate passed directly to thesecond column 11, containing an adsorbent previously equilibrated withthe eluant used to elute the adsorbed material. Use of the same mediumto elute material from the adsorbent in column 5 and equilibrate theadsorbent column 11 avoids the need for timely and involved mediumexchange procedures between the two adsorption steps. Passage of theadsorbed material through column 11 occurs while valve 13 is open andvalve 15 is closed. Eluting adsorbed material from column 11 occurswhile valve 13 is closed and valve 15 is open, thereby passing eluatefrom column 11 directly to a filter (not shown) capable of retainingmaterial having a molecular weight of at least 10,000 daltons. Althoughdemonstrated for use in purifying lactoferrin, the aforesaid method andapparatus is contemplated in other tandem chromatography procedures thatwill be readily apparent to the skilled artisan. For example, theinvention is useful in purifying proteins with similar hydrophobicity tolactoferrin.

[0034] The lactoferrin of the present invention having either no metalloading (iron-free lactoferin, apolactoferrin) or a low metal loading,preferably less than 25%, more preferably less than 20%, most preferablyless than 10% of the metal-binding sites occupied, by virtue of beingcapable of sequestering a significant amount of iron, is useful inapplications to individuals where the removal of iron or othertransition metals from the individual can have beneficial effects, suchas in cosmetics, personal hygiene products, such as feminine douches andmouthwashes, medical and surgical devices and products, topicalantiseptics, ophthalmic solutions, oral and intravenous antibiotics,immunopotentiators, antioxidant, and anti-inflammatory and anti-tumoragents.

[0035] Lactoferrin can be used as an antiseptic in accordance with thepresent invention either alone or in the form of a powder, solution,ointment, aerosol spray, or cream to any part of the subject as an aidin the prevention or treatment of microbial infections. By depriving thesurrounding environment of iron, lactoferrin inhibits the growth ofmicrobes, such as bacteria Preferable antiseptics of the presentinvention include lactoferrin either alone or compounded with carrierssuch as saline, silica, talcum, stearic acid, its magnesium or calciumsalt, polyethyleneglycol, and fatty emulsions and suspensions that willbe readily apparent to the skilled artisan. The lactoferrin ispreferably present in the antiseptic based on 1 ml of the carrier at0.1-2 mg, preferably 0.2-2 mg. An effective amount of the lactoferrinvaries depending on the individual treated, severity of infection, ifany, and the area to which administration is contemplated. Preferably,in treating mammals a twice-daily administration of 0.1-2 mg, morepreferably 0.5-2 mg, most preferably 1 mg, of lactofernin per 1 squarecentimeter effected area is contemplated more preferably 0.1 squarecentimeter. Lactoferrin can be used as an antiseptic in accordance withthe present invention to treat accidental scratches or burns. Forexample, lactoferrin is applied over the affected area in the form of a0.1-2 weight %, preferably 0.1-1 weight % solution, or such a solutionis used to impregnate a Band-Aid type bandage with lactoferrin.Lactoferrin can be used as an antiseptic in accordance with the presentinvention to provide prophylaxis in personal hygiene products. Forexample, the prevention of vaginal infections is accomplished by dailyadministration of 25-50 mg of lactoferrin in a form of douches or pads.Similarly, the oral infections are prevented by daily administration of25-50 mg of lactoferrin in a form of mouthwash. Also, lactoferrinprovides added protection against sexually transmitted infections whencompounded into any device used during sexual activities by either malesor females. For example, lactoferrin is added into lubricant to covercondoms at the concentration of 25-50 mg per application. Lactoferrincan be used as antiseptic in accordance with the present invention toimpregnate any surgical tools, materials or protective clothing that isused by health care personnel. For example, surgical gloves, masks orlinens are covered with a 0.1-2.0% by weight solution of lactoferrin,preferably 0.1-1% by weight. The solution can be applied by a spray,conveniently provided in pressurized aerosol cans or pump bottles.

[0036] Lactoferrin can be useful in the treatment and prevention ofopportunistic bacterial, viral, and fungal infections. Opportunisticinfections are caused by normally non-pathogenic organisms in patientswhose host defense mechanisms have been compromised. By sequesteringiron, lactoferrin inhibits the growth of these organisms, making themmore susceptible to antibiotic therapy. Depending on the type ofinfection involved, treatment can involve one or more types of systemic(oral, nasal, intravenous, etc.) or topical administration. Examples ofsuch infections include pneumonia, acquired immune deficiency syndrome(AIDS), candidiasis, diarrhea, and neonatal sepsis. In treatingpneumonia, for example pneumonia caused by Streptococcus pneumoniae,antibiotics have minimal impact on mortality during the first five daysof illness. By sequestering iron, lactoferrin can inhibit the growth ofpneumococcals, and make them more susceptible to antibiotic therapy.Although administration by oral and intravenous routes is contemplated,a simple delivery system of lactoferrin by inhalation involving topicaladministration to pulmonary membranes is most preferred. Generally,treatment will involve administration three to four times daily of anaqueous solution of lactoferrin in an amount of 100-200 mg per dose fora period of time of 7 to 10 days by inhalation using a known inhaler. Aparticular cause of opportunistic infections is the lowered hostimmunity caused by AIDS. Systemic administration of lactoferrin in AIDSpatients can help prevent or at least delay the onset of secondaryinfections. A variety of treatment modalities are contemplated.Intravenous administration of lactoferrin twice daily in an amount of100-200 mg per injection is recommended for a period of time of one weekfollowed by a one week break. The continuation of this pulse therapy iscontemplated for a period of time of three to six months. Depending onthe particular infection, an additional localized treatment is alsocontemplated. For example, in the case of oral candidiasis, thetreatment will include administration of lactoferrin as a mouthwashtwice daily in an amount of 100-200 mg per dose for a period of time of7 to 10 days. For a pulmonary infection, such as Pneumocistis carinii,the treatment will involve administration of lactoferrin by inhalationfour times daily in an amount of 100-200 mg per dose for a period oftime of 7 to 10 days. For Kaposi's sarcoma treatment will involvetopical administration of lactoferrin in an ointment, twice daily, in anamount of 50-100 mg per dose for three to four weeks. Fungi infections,depending on the type and location, are treated orally, by intravenousinjection, or topical administration. For example, infections, such asvaginal candidiasis, are treated with lactoferrin in a form of douches(vaginal wash) twice daily in an amount of 100-200 mg per dose.Diarrhea, while not usually life threatening, can be dangerous,particularly in infants, because of the potential for fluid imbalance.By virtue of its high affinity for iron, lactoferrin can inhibit thegrowth of pathogens in the gastrointestinal tract. Treatment of diarrheawill involve oral administration of lactoferin twice daily at an amountof 100-200 mg per dose for a period of time of 7 to 10 days. Thetreatment of ulcers caused by Helicobacter pylori is also facilitated bythe use of lactoferrin. Lactoferrin will not only prevent utilization ofiron by the bacteria by sequestering excessive iron from food, it willalso deliver the iron to the small intestine where it is recognized byreceptors specific to lactoferrin. Treatment will involve oraladministration of lactoferrin twice daily in an amount of 200-400 mg perdose for a period of time of 7 to 10 days. Neonatal sepsis, which cancoincide with low production levels of lactoferrin in newborn infants,is also subject to treatment by lactoferrin in accordance with thepresent invention, and particularly in combination with currentantibacterial therapy. Treatment will include intravenous administrationof lactoferrin twice daily in an amount of 100-200 mg per dose for aperiod of time of 7 to 10 days. In patients having burns over a largeportion of the body, the plasma level of lactoferrin increases 10 to 20times the normal amount, the body responding to injury by secreting apowerful antimicrobial agent-lactoferrin. Topical administration oflactoferrin to burn patients, by sequestering iron, will preventdevelopment of surface infection. Treatment will involve topicaladministration of lactofernin in an ointment, cream, or other topicalvehicle twice daily in an amount of 50-100 mg per dose for a period oftime of three to four weeks. Chronic iron overload, known ashemosiderosis, is characterized by greater than normal iron levels incertain body tissues. When associated with tissue injury, the conditionis known as hemochromatosis. Lactoferrin, as the natural chelating agentfor iron, offers a viable treatment for such disorders. Preferabletreatment involves intravenous or subcutaneous doses of lactoferrin oncedaily at an amount of 300-500 mg. Lactofernin can also be used tosequester iron implicated in heart disease. By sequestering iron thatpromotes the oxidation of lipids, which when oxidized can clog arteries,lactoferrin can aid in reducing heart attacks. The prophylactictreatment involves intravenous administration of lactoferrin twiceweekly in an amount of 200-500 mg. The treatment is contemplated forhigh risk patients having high levels of cholesterol. Ischemic heartdisease remains the most important cause of morbidity and mortality inthe U.S. Over the last decade acute revascularization with thrombolyticdrugs has emerged as the standard treatment for patients with acutemyocardial infraction. Considerable evidence has emerged over the lastdecade which indicates that iron may play a key role in pathogenesis ofreperfusion injury in the heart. Lactoferrin can sequester large amountsof iron that tend to be released following heart attacks, which thusreduces the amount of iron available for reacting with oxygen togenerate free radicals, which cause damage to muscle fibers and cellwalls. Preferable treatment involves intravenous administration oflactoferrin immediately after heart attack in an amount of 500-1,000 mg.The treatment of a pneumococcal, streptococcal or staphylococcalinfection following trauma of the cornea is also facilitated bylactoferrin. These infections are usually primary causes of cornealulcers. The treatment will involve topical administration of lactoferrinin an ointment, eye drops or other topical vehicle twice daily in anamount of 10-20 mg per application. Lactoferrin can also be utilized tosequester iron from contact lenses having an application in antiseptictreatment of lenses between wearing times. The effective solution willconsist of 0.1-1.0% of lactoferrin in water. The treatment of tumors,such as brain tumors, can also be facilitated by the use of lactoferrin.Newly developed catherization procedures permit the delivery oflactoferrin directly to a blood supply aorta of a tumor, such as a braintumor, which, by reducing the iron necessary for metabolism of the tumorcells, can prevent the growth of the tumor. Treatment will involveweekly administration of lactoferrin through a microcatheter in anamount of 1-2 grams per dose for a period of time of three to sixmonths. Lactoferrin can also be used as an adjuvant in vaccination.Subunit antigens and peptides made by recombinant DNA technology are notvery immunogenic, making their use as vaccines contingent upon theavailability of adjuvants that are safe for use in humans and are ableto augment sufficiently the immunogenicity of these molecules. Becauseit modulates a number of immunological responses including myeloiddifferentiation, modulation of macrophage-mediated cytotoxicity, andregulation of the primary antibody response, lactoferrin can be used assuch an adjuvant. The lactoferrin is systemically administered at100-200 mg per vaccination. Lactoferrin administration before, during,or after vaccination is contemplated based on the specific antigen usedfor the vaccine.

[0037] The nutritional supplement of the present invention contains aneffective amount of lactoferrin loaded with iron, either alone or incombination with one or more nutritionally acceptable carriers oradjuvants. Preferred nutritional supplements include tablets, gelatincapsules, or liquids containing the lactoferrin together with adjuvantsor diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol,cellulose, and glycine; binders, such as magnesium aluminum silicate,starch, paste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, and polyvinylpyrrolidone; disintegrants, such asstarches, agar, alginic acid or its sodium salt, and effervescentmixtures; as well as absorbents, colorants, flavors and sweeteners.Alternatively, the iron-loaded lactoferrin can be added to foods such asbaby formula, cereal, and ice cream to enhance the nutritional value ofthe food. The preferred amount of iron-loaded lactofernin in thesupplement based on the weight of 1 g of the supplement is 5-50 mg, morepreferably 20-30 mg, and most preferably 25 mg. An effective dailyamount of based on the individual, iron-loaded lactoferrin varies, fromabout 10-30 mg, preferably 20-30 mg, and more preferably 25 mg. Loadinglactoferrin with iron is accomplished by simple titration with, e.g.,ferrous ammonium in the presence of bicarbonate, according to methodsthat will be readily apparent to the skilled artisan. Preferred loadingis such that at least about 35%, more preferably at least about 50%, andmost preferably at least about 70%, of the metal binding sites are ironbound. Lactoferrin can be applied to food (either solid or liquid) toretard spoilage in accordance with the present invention either alone orcompounded with any of the aforesaid nutritionally acceptable carriersor diluents. By sequestering iron, and thereby suppressing its catalyticactivity, the lactoferrin reduces the iron available for eithermicrobial multiplication or the production of potentially cell-damagingfree-radicals that are formed in iron catalyzed reactions from hydrogenperoxide or superoxide. For example, the lactoferrin is particularlyuseful in inhibiting rancidity in meat, which is iron-dependent lipidperoxidation. To inhibit microbial growth, particularly in liquid foodssuch as beer and wine, the lactoferrin can be added directly to theliquid or used to coat filters through which the liquid food passesduring processing. An effective amount of the lactoferrin for retardingspoilage varies depending on the type and amount of food contemplated.Preferably, the amount of lactoferrin applied to food in accordance withthe present invention varies from 0.1-1 mg/ml of food with which it ismixed, or based on the surface area of the filter or solid food to whichit is applied from 0.1-1 mg/cm². The preferred amount of lactoferrincompounded with a carrier in a food additive for retarding spoilagevaries based on 1 ml of the carrier from 0.1-2 mg, preferably 0.2-2 mg.

[0038] The antiseptic, dietary supplement, and food-spoilage retardantof the present invention can be sterilized and/or contain adjuvants,such as preserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. Said compositions are prepared according to conventionalmixing, granulating, and coating methods.

[0039] The lactoferrin contemplated for use in accordance with thepresent invention is preferably of human origin, more preferably via DNArecombinant means, but other lactoferrins, such as natural bovine, goatand porcine lactoferrin, isolated and purified using methods applicableto natural human lactoferrin, are contemplated.

[0040] The following non-limiting examples are provided to illustratethe present invention. All parts and percentages are by weight unlessindicated otherwise.

EXAMPLE 1 Cloning and Expression of Lactoferrin

[0041] Human lactoferrin is obtained from a genetically alteredorganism. Using breast tissue excised during the mastectomy of a womanduring the eighth month of pregnancy, a human mammary gland genomiclibrary is prepared according to the procedure of Gubler, et al., Gen.,40, 1-8 (1983), which disclosure is hereby incorporated by reference. Ahuman mammary gland genomic library of cDNA ligated to λgt11 isavailable from Clontech, Calif. The library is transferred onto agarplates containing a high density of E. coli Y 1090 (Clontech, Calif.)(5×10⁴ plaques per 90 mm plate or 1.4×10⁵ plaques per 150 mm plate). Theplates are allowed to stand for 3.5 hours at 42° C. to obtain a lyticgrowth of the phage. The plates are thereafter overlaid withnitrocellulose filters (Schleiher and Schnell Inc., Wouburn, Mass.,under no. BA 85 NC) and heated in an incubator at 37° C. for 3.5 hours.

[0042] Positive clones (i.e., containing the cDNA) are identified on themembranes using rabbit antibody to natural human lactoferrin purified inaccordance with Example 9 described hereafter. Nitrocellulose filtersare removed from the plates after plaque transferral, and coated withthe antibody purified as described under Example 9, which hybridizeswith positive plaques. Following removal of excess antibody, positiveplaques are developed by first applying an anti-rabbit IgG conjugatedwith biotin (Sigma Chemical Co., St. Louis, Mo.), and then, followingremoval of excess biotin conjugate, applying avidin conjugated withhorse radish peroxidase (Sigma Chemical Co., St. Louis, Mo.). Finally,the positive plaques are identified in the reaction catalyzed by horseradish peroxidase using as an enzyme substrate 4chloro-1-Naphtol.

[0043] The positive plaques are then used to infect E. coli Y 1090 toproduce large amounts of phage in accordance with procedures set forthin Davis, et. al. (1986), supra. The resulting bacteriophage is purifiedusing 10% polyethylene glycol and DNA is isolated from the phageaccording to the procedures disclosed in Kislow, N. A. R., 14, 6767(1986), which disclosure is hereby incorporated by reference. Followingthe procedures in Davis, et al. (1986), supra, the cDNA insert encodinglactoferrin is sub-cloned as follows: the cDNA insert is cut out fromthe phage DNA using EcoR I and purified using a high resolutionion-exchange chromatography column (GEN-PAK™ Fax, Millipore Corporation,Waters Chromatography Division, Milford, Mass.). The thus purified cDNAinsert is ligated using T4 DNA ligase into plasmid pGEM-4 (Promega,Madison, Wis.) as described by Yanish-Perron et al., Gen., 33, 103-109(1985), which disclosure is hereby incorporated by reference that hasbeen cut using EcoR I using standard techniques. The plasmid containingthe insert is then transferred into E. coli JM109 (Promega, Madison,Wis.) as described by Hanahan, J. Mol. Blot, 166, 557 (1983), whichdisclosure is hereby incorporated by reference. The bacteria aretransferred to agar plates containing ampicillin and the positivecolonies grown. The plasmid is then isolated and the cDNA insert is cutfrom the plasmid using EcoR I and purified by ion exchangechromatography as described above.

[0044] The cDNA insert is then ligated into the Pichia pastorisexpression vector pAO804, so as to be flanked by the 5′ and 3′regulatory sequences of the methanol-induced alcohol oxidase gene (AOX1)of P. pastoris in accordance with the procedures described bySreekrishna, et al., Biochemistry, 28, 4117-4125 (1989); and Rothstein,Methods in Enzymology, 101, 202-210 (1983), which disclosures are herebyincorporated by reference. The resulting vector is then transformed intoPichia pastoris GTS115 (His4) by the method of spheroplast as describedby Creeg, et al., Mol. Cell. Biol., 5, 3376-3385 (1985), whichdisclosure is hereby incorporated by reference. The selectedtransformant cells are grown in 10 ml buffered minimal glycerol-complexmedium at 30° C. with shaking for three days. The saturated culture iscentrifuged for 10 minutes at 3000 G. The cell pellet is resuspendedwith 2 ml of buffered minimal methanol-complex medium and returned tothe 30° C. shaker for another three days. Cells are pelleted bycentrifugation and both the pellet and supernatant are analyzed for thepresence of lactoferin. Alternatively, the procedure of Hagenson et al.,Enzyme Microb. Technol., 11, 650-656 (1989), which disclosure is herebyincorporated by reference, is followed to grow the cells in a fermentorup to OD₆₀₀ of about 1.0 then harvested, and washed with and suspendedin minimal methanol media at an OD₆₀₀ about 4.0. The culture is held at30° C. while maintained at a pH of 5.0 by adding NH₃ gas to the airstream. Expressed lactoferrin is recovered from the supernatant of thefermentation media following 15 minutes centrifugation at 5000 rpm usinga Beckman J-21B with a Rotor JA 14.

EXAMPLE 2 Concentration and initial Purification of Lactoferrin

[0045] One liter of the supernatant from Example 1 is adjusted to about40C. and filtered under pressure through a polysulfone ultrafiltrationmembrane having a pH operating range of 1-14 on a polypropylene meshsupport (PELLICON™ Cassette filter System assembled with Procon pump andPTGC membrane, Millipore Corporation, Milford, Mass.) to retain proteinsin excess of about 10,000 molecular weight. Pressure with simultaneouscirculation is applied until 900 ml of ultra filtrate is collected. Aflow rate of about 100 ml per minute is maintained during the filtrationprocess. The retained material (100 ml) is diluted with 900 ml 20 mMphosphate buffer (pH 7.4) and re-filtered, which is repeated four times(final exchange ratio=10,000). The final material retained is sterilized(0.22 micron GELMAN™ filter).

EXAMPLE 3 Purification of Lactoferrin using Affinity Chromatography

[0046] Human lactoferrin is purified using affinity chromatography inwhich the affinity ligand is the reactive dye Cibacron blue F3G-A. Thesterilized material obtained in Example 2 is adjusted to a pH of 7.5 anda final concentration of sodium chloride of 0.5 M. This material is thenapplied onto a column (5 cm×35 cm) packed with cross-linked agarosecoupled to the dye (Pharmacia Fine Chemicals, Uppsala, Sweden, BLUESEPHAROSE™ CL-6B) and previously equilibrated with 50 mMN-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) buffer(pH 7.5) containing 0.5 M sodium chloride. Adsorption is performed at aflow rate of 1 ml/min followed by washing the column with 2 bed volumesof the same HEPES buffer. The non-adsorbed fraction is discarded and theadsorbed fraction containing lactoferrin is eluted from the column bedusing 2 bed volumes of 50 mM HEPES buffer (pH 7.5) containing 1 M sodiumchloride.

EXAMPLE 4 Purification of Lactoferrin using Control Pore Glass (CPG)Chromatography

[0047] Human lactoferrin is purified using control pore glass (CPG)chromatography. The eluate from Example 3 is applied onto a column (1.2cm×10 cm) packed with CPG beads (CPG 00350, ElectroNucleonics,Fairfield, N.J.) and previously equilibrated with 50 mM HEPES buffer (pH7.5) containing 1 M sodium chloride. Adsorption is performed at a flowrate of 1 ml/min followed by washing the column with 2 bed volumes ofthe same buffer. The non-adsorbed fraction is discarded, and theadsorbed fraction is eluted with 2 bed volumes of 0.25 Mtetramethylammonium chloride (TMAC, pH 7.5). The eluate is filtered on amembrane capable of excluding material having a molecular weight greaterthan 10,000 daltons (AMICON™ YM 10). The filtered material is thensterilized (0.22 micron GELMAN™ filter) and frozen at −200C.

EXAMPLE 5 Purification of Lactoferrin using Immobilized Nickel IonAffinity Chromatography

[0048] Human lactoferrin is purified using immobilized metal ionaffinity chromatography (IMAC). An imminodiacetic acid-epoxy activatedgel (Pharmacia Fine Chemicals, Uppsala, Sweden, CHELATING SEPHAROSE™ 6B)is washed with water and equilibrated with 0.1 M sodium acetate buffer(pH 4.0) containing 1 M sodium chloride. The gel is then packed into achromatographic column (1.2 cm×10 cm) and saturated with 4 bed volumesof the same sodium acetate buffer further containing 5 mg/ml of nickelchloride. Excess metal is washed from the column with the sodium acetatebuffer, and the gel is equilibrated with 20 mM HEPES buffer (pH 7.0)containing 1 M sodium chloride and 2 mM imidazole.

[0049] The product of Example 4 is mixed with HEPES, sodium chloride,and imidazole to obtain a pH of 7.0, 20 mM HEPES, 1 M sodium chloride,and 2 mM imidazole. The mixture is applied onto the column at a flowrate of about 1 ml/min followed by,washing the gel with 2 bed volumes of20 mM HEPES buffer (pH 7.0) containing 1 M sodium chloride and 2 mMimidazole. The non-adsorbed fraction is discarded, and the adsorbedfraction containing lactoferrin is eluted using 2 bed volumes of 20 mMHEPES buffer (pH 7.0) containing 1 M sodium chloride and 20 mMimidazole.

EXAMPLE 6 Purification of Lactoferrin using Immobilized Copper IonAffinity Chromatography

[0050] Human lactoferrin is purified using immobilized metal ionaffinity chromatography (IMAC). An imminodiacetic acid-epoxy activatedgel (Pharmacia Fine Chemicals, Uppsala, Sweden, CHELATING SEPHAROSE™ 6B)is washed with water and equilibrated with 0.1 M sodium acetate buffer(pH 4.0) containing 1 M sodium chloride. The gel is then packed into achromatographic column (1.2 cm×10 cm) and saturated with 4 bed volumesof the same sodium acetate buffer further containing 5 mg/ml of coppersulfate. Excess metal is washed from the column with the sodium acetatebuffer, and the gel is equilibrated with 50 mM TRIS-HCL buffer (pH 8.0)containing 1 M sodium chloride.

[0051] Lactoferrin equilibrated to 50 mM TRIS-HCL pH 8.0, 1 M NaCl isapplied onto the column at a flow rate of about 1 ml/min followedby,washing the gel with 2 bed volumes of 50 mM TRIS-HCL buffer (pH 7.0)containing 1 M sodium chloride. The non-adsorbed fraction is discarded,and the adsorbed fraction containing lactoferrin is eluted using 2 bedvolumes of 200 mM sodium acetate buffer pH 3.0.

EXAMPLE 7 Purification of Lactoferrin Using T-Gel AffinityChromatography

[0052] Human lactoferrin is purified using T-gel affinitychromatography. T-gel adsorbent is prepared according to the proceduredescribed by Porath, et al., Methods in Enzymology, 44, 19-45 (1976),which disclosure is hereby incorporated by reference, and packed into acolumn (1.2 cm×10 cm). The final product of Example 4 is adjusted to apH of 7.5 and a final concentration as follows: 50 mM PIPES buffer(piperazine-N,N′-bis[2-ethanesulfonicacid] and1,4-piperazinediethanesulfonicacid) buffer and 0.7 M ammonium sulfate.The adjusted material is applied on the column that has been previouslyequilibrated to 50 mM PIPES buffer (pH 7.5) containing 0.7 M ammoniumsulfate with a flow rate of about 1 ml/min. The non-adsorbed fractioncontaining lactoferrin is adjusted to a concentration of 0.1 M ammoniumsulfate and then applied to an identical T-gel column previouslyequilibrated to 50 mM PIPES buffer (pH 7.5) containing 1.0 M ammoniumsulfate. The column is then washed with 7-8 bed volumes of 50 mM PIPESbuffer (pH 7.5) containing 1.0 M ammonium sulfate, with lactoferrinbeing present in the non-adsorbed fraction.

EXAMPLE 8 Purification of Lactoferrin Using Hydrophobic InteractionChromatography

[0053] Human lactoferrin is purified using hydrophobic interactionchromatography on a cross-linked agarose gel coupled to phenyl glycidylether (PHENYL SEPHAROSE™ CL-4B, Pharmacia Fine Chemicals, Uppsala,Sweden). The gel is packed into a column and equilibrated to 50 mM PIPESbuffer (pH 7.0) containing 1 M ammonium sulfate. The product of Example4 is adjusted to the equilibrating buffer and applied onto the column ata flow rate of 1 ml/min. The non-adsorbed fraction is discarded and theadsorbed fraction containing lactoferrin is eluted using 2 bed volumesof 50 mM PIPES buffer (pH 7.0).

EXAMPLE 9 Purification of Anti-Lactoferrin Serum

[0054] Anti-lactoferrin serum is purified by affinity chromatography.The adsorbent substrate for affinity chromatography is prepared bycyanogen bromide activation as described by Axen et al., Nature, 214,1302-1304 (1967), which disclosure is hereby incorporated by reference.The substrate (Pharmacia Fine Chemicals, Uppsala, Sweden,CNBR-SEPHAROSE™ 4B) is coupled to human lactoferrin, which acts as theaffinity ligand, as follows. One gram of substrate is swelled with 1 mMHCl and washed with the same solvent on a sintered glass filter. Ten mgof natural human lactoferin (Sigma Chemical Co., St. Louis, Mo.) isdissolved in 0.1 M NaHCO₃ buffer (pH 8.3) containing 0.5 M sodiumchloride (coupling buffer). The resulting solution is mixed with thewashed substrate gel for 2 hours, and then mixed with 0.2 M glycinebuffer (pH 4.0) for 2 hours. The gel is then washed with couplingbuffer, followed by 0.1 M acetate buffer (pH 4.0) containing 0.5 Msodium chloride, followed again by coupling buffer to form theadsorbent. The adsorbent is packed into a column and washed with 20 mMphosphate buffer (pH 7.4) containing 0.5 M sodium chloride.Anti-lactoferrin serum obtained from an inoculated rabbit (SigmaChemical Co., St. Louis, Mo.) is passed through the column at a flowrate of 1 ml/min and the non-adsorbed material discarded. Adsorbedmaterial containing the purified protein is eluted with 2 bed volumes of0.2 M glycine buffer (pH 2.0) containing 0.5 M of sodium chloride. Theeluate is neutralized with 0.1N NaOH to obtain pH 7.5 and thensterilized (0.22 micron GELMAN™ filter) and frozen at −20° C.

EXAMPLE 10 Cloning of Human Lactoferrin Gene by PCR

[0055] The human lactoferrin gene is cloned by PCR amplification from ahuman mammary gland library in λgt11. Phage DNA is prepared from thelibrary by using standard procedures described in Maniatis (1989),supra. Oligonucleotide primers are prepared to the 5′- and 3′-ends ofthe gene based on the sequence published by Powell, et al. (1990),supra. The 5′-oligo is designed to begin at the coding sequence for thefirst glycine of the mature lactoferrin protein as defined by Powell, etal. (1990), supra, and the 3′-oligo included the lactoferrin stop codonand 21 nucleotides beyond. These oligonucleotides are prepared on anApplied Biosystems DNA synthesizer Model 380A and were obtained fromRoswell Park Cancer Institute, Buffalo, N.Y. Each oligo also created arestriction endonuclease recognition site, the 5′-oligo contained a BamHI site and the 3′-oligo an Xba I site. The sequences of theseoligonucleotides are given below:

[0056] 5′-oligo AGCGGATCCGGCCGTAGGAGAAGGAGTGTTCAGTGG (Seq. ID No.3) BamHI

[0057] 3′-oligo CGATCTAGATTACTTCCTGAGGAATCCACAGGC (Seq. ID No.4) Xba I

[0058] PCR amplification of the cDNA library using these oligonucleotideprimers is performed with a temperature cycler (Genetic Thermal Cycler,Precision Scientific), utilizing Tag polymerase. Amplificationconditions are: 1.5 min. 94° C., 2 min. 50° C., 5 min. 60° C. for 35successive cycles. PCR amplification of the cDNA library resulted inproduction of a 2 kbp product, which is not observed when either primeris used alone, as shown by agarose gel analysis in FIG. 4 (molecularmass markers are indicated at the left of the figure).

EXAMPLE 11 Restriction Map and Sequencing of Lactoferrin Gene

[0059] The PCR amplified fragment is cut with restriction enzymes BamH Iand Xba I and following agarose gel purification is ligated into plasmidpUC 118 and transformed into E. coli JM101. A large amount of plasmidDNA is prepared for restriction enzyme digestion and sequencing. ThecDNA lactoferrin insert is cut out with BamH I and Xba I and afteragarose gel purification is subjected to digestion with the followingrestriction enzymes: Bgl I, HgiA I, Pvu II, and Stu I. The digestion iscarried out at 37° C. for two hours. Size of the DNA fragments isdetermined in 2% agarose gel and is shown here as FIG. 8. FIG. 8 is arestriction fragment map of the cDNA lactoferrin gene. The top diagramshows the predicted digestion pattern, while the bottom gel illustratesthe size of fragments obtained during digestion. Lanes 1 and 7 showmolecular mass markers, lane 3 shows the insert encoding the lactoferringene, and lanes 2, 4, 5, and 6 show digestion of the insert withrestriction of the enzymes Bgl I, HgiA I, Pvu II and Stu I,respectively. The digestion of cDNA lactofernin with restriction enzymesBgl I, HgiA I and Pvu II generate fragments which follow the pattern ofa full length lactoferrin clone. Digestion with Stu I generates two(rather than the predicted three) fragments due to methylation of theDNA sequences which overlap the recognition sequence of the restrictionendonuclease.

[0060] The cDNA insert encoding the human lactoferrin gene is sequencedin multiple directions using the dideoxy termination method of Sanger,Proc. Nat. Acad. Sci. USA, 74, 5463-5467 (1977), which disclosure ishereby incorporated by reference. Oligomers of 18 bases corresponding tothe sequence of 250, 502, 751, 1003, 1252, 1502, and 1751 residues weremade and used as primers. The nucleotide sequence (Seq. ID No. 1) of theinstant clone is shown in FIG. 3.

EXAMPLE 12 Construction of Plasmids pUC118-LF and pUC118-LFS

[0061] The PCR product of Example 10 is digested with BamH I and Xba I,isolated from a 0.7% low melting agarose and ligated into pBlueScriptKS+(Strata gene Corp.), which is digested with the same restrictionenzymes. The resulting plasmid is designated pBSlacto. The lactoferringene containing fragment is then cut out from pBSlacto using Hind IIIand Sst I and subcloned into pUC118, which is also cut with theseenzymes. The resulting plasmid is named pUC118-LF and contains themature lactoferrin gene, that is the nucleotide sequence (Seq. ID No. 1,FIG. 3) encoding the mature lactoferrin protein (Seq. ID No. 2, FIG. 3).

[0062] To allow secretion of the lactoferrin protein, the signalsequence is added to the 5′-end of the clone using syntheticoligonucleotides based on the signal sequence published by Powell, etal. (1990), supra. The oligonucleotides used are shown below:

[0063] Lacto signal I: AAGCTTATGAAACTTGTCTTCCTCGTCCTGTTCTTCCTCGGG HindIII (Seq. ID No. 5)

[0064] Lacto signal II: GATCCAGCCAGAGAGAGTCCGAGGGCCCCGAGGAA BamHI (Seq.ID No. 6)

[0065] The Lacto signal I contains a Hind III restriction site and thefirst 12 codons including the initial methionine. Lacto signal IIcontains a BamH I restriction site and the final 10 codons of the signalpeptide sequence. The final 9 nucleotides of two oligos arecomplementary and are annealed, filled-in with the Klenow fragment ofDNA polymerase I from E. coli. The resulting double-stranded DNAfragment is digested with Hind III and BamH I and ligated into pUC118-LF, which is similarly digested. The resulting plasmid, pUC118-LFS,contains a signal sequence with the natural lactoferrin sequence.

EXAMPLE 13 Construction of Expression Plasmid pHIL-D1-LFS

[0066] Plasmid pUC118-LFS of Example 12 is digested with Hind III andXba I and the digested DNA is filled in with Klenow DNA polymerase toproduce blunt ends. The LF gene with the signal sequence (LFS) is gelpurified and ligated to pHIL-DI cut with EcoR I and blunt ended.pHILD1-LFS with the correct orientation is identified by screening quickplasmid DNA preps for correct orientation by Stu I digestion. In thecorrect orientation fragments of size 3.856 Kb and 5.84 Kb are expected.In the wrong orientation fragments of sizes 2.23 Kb and 7.46 Kb areexpected. Several clones (>12) in the correct orientation are obtained,pooled, and used for Pichia transformation. In pHILD1-LFS the 5′untranslated region is as follows: . . . ATTATTCGAAACGAGGAATTAGCTTATG(Seq. ID No. 7). The nucleotide composition of −1 to −25 position isAT:GC=68:32, which is in the preferred range for Pichia expression.

[0067] Plasmid pHILD1-LFS is divided into two portions. One part iscleaved with Sac I (to direct integration into the AOX1 locus of Pichiastrain KM71 [aox1::ARG4, His4]) and the other part is cleaved with Sal I(to direct integration into His4 locus of KM71). Approximately 240 His+transformants are obtained with Sac I cut DNA, and approximately 120His+transformants are obtained with Sal I cut DNA.

EXAMPLE 14 Construction of Expression Plasmid pPIC9-LF using AlphaMating Factor AMF

[0068] Plasmid pPIC9 (Phillips Petroleum Co., Bartlesville, Okla.) iscut with Not I restriction enzyme and alkaline phosphatase treated. Nextit is cut with Xho I restriction enzyme. The vector fragment is purifiedon agarose gel to separate it from the small Aho I-Not I fragment(approx. 43 bp). The purified vector is ligated with alpha matingfactor, which is the following 11-mer synthetic oligonucleotide, inwhich only the top strand is kinased.

[0069] (P-TCGAGAAAAGA CTTTTCTCCGG-OH) (Seq. ID No. 8)

[0070] The pPIC9 vector fragment linked with the 11-mer oligonucleotideis gel purified to separate it from excess 11-mer oligonucleotides. Theresulting gel-purified linked vector fragment, which has Xma III and NotI ends, is ligated with the gel-purified Xma III fragment containing themature lactoferrin gene from Example 13 (Xma III end is compatible withNot I end, thus the Xma III fragment can ligate into the Xma III/Not Iends of the vector). The resulting vector is transformed into Pichiapastoris GTS 115 (His4) (Phillips Petroleum Co., Bartlesville, Okla.) bythe method of spheroplast. The selected transformant cells are used forexpression of lactoferrin in a shake flask experiment.

EXAMPLE 15 Expression Purification and Characteriztion of HumanLactoferrin from Pichia pastoris

[0071] The selected transformant cells from Example 14 are grown tosaturation in 10 ml of buffered minimal glycerol-complex medium in a 50ml plastic tube in a 30° C. shaker at 300 revolutions/min. for threedays. The saturated culture is centrifuged for 10 minutes at 3,000 G.The cell pellet is resuspended with 2 ml of buffered minimal methanolcomplex medium and returned to the 30° C. shaker for another three days.The supernatant is analyzed for presence of lactoferrin by SDS-PAGE andWestern Blot. Lactoferrin is isolated and concentrated from the growthmedia in one step chromatography. Epoxy agarose is saturated withcopper-ions (CuSO_(4,) 5 mg/ml), washed with 50 mM Tris-HCl buffer, pH8.0. About 200 mg of gel is used for adsorption of lactoferrin from 1 mlof expression medium. The gel is washed with 10 ml of equilibrationbuffer and lactoferrin is recovered from the gel with 1 ml of 0.2 Mammonium-acetate buffer, pH 3.0.

[0072] After lyophilization, proteins are dissolved in 50 ml of SDSsample buffer. SDS-PAGE electrophoresis is performed on 9% acrylamidegels according to Laemmli, U.K., (1970) Nature 227, 680-685, thedisclosure of which is incorporated by reference herein, and silverstained. After SDS-PAGE electrophoresis, proteins are transferred tonitrocellulose filters with semi-dry transferring apparatus. The filtersare blocked overnight with 5% non-fat dry milk in 50 mM Tris HCl buffer,pH 8.0. After a brief washing with 50 mM Tris-HCl buffer, pH 8.0,filters are incubated with rabbit monospecific polyclonalanti-lactoferrin antibodies (1:20,000 dilution) for 2 hr at roomtemperature, washed four times with 50 mM Tris-HCl, 0.15 M NaCl, 0.05%Tween buffer, pH 8.0, and incubated for 30 min. with protein Aconjugated to a horseradish peroxidase. The washes are repeated; thefilters are incubated with a chemiluminescent detection kit for 1 min.and exposed to Kodak X-ray film for 1 min.

[0073]FIG. 9 shows the SDS-PAGE analysis (elution from Cu⁺⁺-epoxyagarose) of the thus recovered lactoferrin in lane 4 compared to nativelactoferrin from human milk as a positive control in lane 2 and anegative control in lane 3 (medium pass through Cu⁺⁺-epoxy agarose).FIG. 10 shows the western blot analysis (elution from Cu⁺⁺-epoxyagarose) of the thus recovered lactoferrin in lane 3 compared to nativelactoferrin from human milk as a positive control in lane 1and anegative control in lane 2 (medium pass through Cu⁺⁺-epoxy agarose).Molecular mass markers (in kDa) are indicated at the left of FIGS. 9 and10. The results show that the expressed

1 8 2086 base pairs nucleic acid single linear DNA (genomic) NO NO CDS1..2086 1 GGA TCC GGC CGT AGG AGA AGG AGT GTT CAG TGG TGC GCC GTA TCCCAA 48 Gly Ser Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln 15 10 15 CCC GAG GCC ACA AAA TGC TTC CAA TGG CAA AGG AAT ATG AGA AAA GTG96 Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val 20 2530 CGT GGC CCT CCT GTC AGC TGC ATA AAG AGA GAC TCC CCC ATC CAG TGT 144Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys 35 40 45ATC CAG GCC ATT GCG GAA AAC AGG GCC GAT GCT GTG ACC CTT GAT GGT 192 IleGln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly 50 55 60 GGTTTC ATA TAC GAG GCA GGC CTG GCC CCC TAC AAA CTG CGA CCT GTA 240 Gly PheIle Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val 65 70 75 80 GCGGCG GAA GTC TAC GGG ACC GAA AGA CAG CCA CGA ACT CAC TAT TAT 288 Ala AlaGlu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr 85 90 95 GCC GTGGCT GTG GTG AAG AAG GGC GGC AGC TTT CAG CTG AAC GAA CTG 336 Ala Val AlaVal Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu 100 105 110 CAA GGTCTG AAG TCC TGC CAC ACA GGC CTT CGC AGG ACC GCT GGA TGG 384 Gln Gly LeuLys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp 115 120 125 AAT GTCCCT ATA GGG ACA CTT CGT CCA TTC TTG AAT TGG ACG GGT CCA 432 Asn Val ProIle Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro 130 135 140 CCT GAGCCC ATT GAG GCA GCT GTG GCC AGG TTC TTC TCA GCC AGC TGT 480 Pro Glu ProIle Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys 145 150 155 160 GTTCCC GGT GCA GAT AAA GGA CAG TTC CCC AAC CTG TGT CGC CTG TGT 528 Val ProGly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu Cys 165 170 175 GCGGGG ACA GGG GAA AAC AAA TGT GCC TTC TCC TCC CAG GAA CCG TAC 576 Ala GlyThr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr 180 185 190 TTCAGC TAC TCT GGT GCC TTC AAG TGT CTG AGA GAC GGG GCT GGA GAC 624 Phe SerTyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp 195 200 205 GTGGCT TTT ATC AGA GAG AGC ACA GTG TTT GAG GAC CTG TCA GAC GAG 672 Val AlaPhe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu 210 215 220 GCTGAA AGG GAC GAG TAT GAG TTA CTC TGC CCA GAC AAC ACT CGG AAG 720 Ala GluArg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys 225 230 235 240CCA GTG GAC AAG TTC AAA GAC TGC CAT CTG GCC CGG GTC CCT TCT CAT 768 ProVal Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His 245 250 255GCC GTT GTG GCA CGA AGT GTG AAT GGC AAG GAG GAT GCC ATC TGG AAT 816 AlaVal Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp Asn 260 265 270CTT CTC CGC CAG GCA CAG GAA AAG TTT GGA AAG GAC AAG TCA CCG AAA 864 LeuLeu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys 275 280 285TTC CAG CTC TTT GGC TCC CCT AGT GGG CAG AAA GAT CTG CTG TTC AAG 912 PheGln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys 290 295 300GAC TCT GCC ATT GGG TTT TCG AGG GTG CCC CCG AGG ATA GAT TCT GGG 960 AspSer Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly 305 310 315320 CTG TAC CTT GGC TCC GGC TAC TTC ACT GCC ATC CAG AAC TTG AGG AAA 1008Leu Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys 325 330335 AGT GAG GAG GAA GTG GCT GCC CGG CGT GCG CGG GTC GTG TGG TGT GCG 1056Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala 340 345350 GTG GGC GAG CAG GAG CTG CGC AAG TGT AAC CAG TGG AGT GGC TTG AGC 1104Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser 355 360365 GAA GGC AGC GTG ACC TGC TCC TCG GCC TCC ACC ACA GAG GAC TGC ATC 1152Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile 370 375380 GCC CTG GTG CTG AAA GGA GAA GCT GAT GCC ATG AGT TTG GAT GGA GGA 1200Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 385 390395 400 TAT GTG TAC ACT GCA GGC AAA TGT GGT TTG GTG CCT GTC CTG GCA GAG1248 Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu 405410 415 AAC TAC AAA TCC CAA CAA AGC AGT GAC CCT GAT CCT AAC TGT GTG GAT1296 Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp 420425 430 AGA CCT GTG GAA GGA TAT CTT GCT GTG GCG GTG GTT AGG AGA TCA GAC1344 Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp 435440 445 ACT AGC CTT ACC TGG AAC TCT GTG AAA GGC AAG AAG TCC TGC CAC ACC1392 Thr Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr 450455 460 GCC GTG GAC AGG ACT GCA GGC TGG AAT ATC CCC ATG GGC CTG CTC TTC1440 Ala Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe 465470 475 480 AAC CAG ACG GGC TCC TGC AAA TTT GAT GAA TAT TTC AGT CAA AGCTGT 1488 Asn Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser Cys485 490 495 GCC CCT GGG TCT GAC CCG AGA TCT AAT CTC TGT GCT CTG TGT ATTGGC 1536 Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile Gly500 505 510 GAC GAG CAG GGT GAG AAT AAG TGC GTG CCC AAC AGC AAC GAG AGATAC 1584 Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr515 520 525 TAC GGC TAC ACT GGG GCT TTC CGG TGC CTG GCT GAG AAT GCT GGAGAC 1632 Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp530 535 540 GTT GCA TTT GTG AAA GAT GTC ACT GTC TTG CAG AAC ACT GAT GGAAAT 1680 Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn545 550 555 560 AAC AAT GAG GCA TGG GCT AAG GAT TTG AAG CTG GCA GAC TTTGCG CTG 1728 Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe AlaLeu 565 570 575 CTG TGC CTC GAT GGC AAA CGG AAG CCT GTG ACT GAG GCT AGAAGC TGC 1776 Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg SerCys 580 585 590 CAT CTT GCC ATG GCC CCG AAT CAT GCC GTG GTG TCT CGG ATGGAT AAG 1824 His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met AspLys 595 600 605 GTG GAA CGC CTG AAA CAG GTG TTG CTC CAC CAA CAG GCT AAATTT GGG 1872 Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys PheGly 610 615 620 AGA AAT GGA TCT GAC TGC CCG GAC AAG TTT TGC TTA TTC CAGTCT GAA 1920 Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln SerGlu 625 630 635 640 ACC AAA AAC CTT CTG TTC AAT GAC AAC ACT GAG TGT CTGGCC AGA CTC 1968 Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu AlaArg Leu 645 650 655 CAT GGC AAA ACA ACA TAT GAA AAA TAT TTG GGA CCA CAGTAT GTC GCA 2016 His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln TyrVal Ala 660 665 670 GGC ATT ACT AAT CTG AAA AAG TGC TCA ACC TCC CCC CTCCTG GAA GCC 2064 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu LeuGlu Ala 675 680 685 TGT GAA TTC CTC AGG AAG TAA A 2086 Cys Glu Phe LeuArg Lys * 690 695 694 amino acids amino acid linear protein 2 Gly SerGly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln 1 5 10 15 ProGlu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val 20 25 30 ArgGly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys 35 40 45 IleGln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly 50 55 60 GlyPhe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val 65 70 75 80Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr 85 90 95Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu 100 105110 Gln Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp 115120 125 Asn Val Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro130 135 140 Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala SerCys 145 150 155 160 Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu CysArg Leu Cys 165 170 175 Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser SerGln Glu Pro Tyr 180 185 190 Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu ArgAsp Gly Ala Gly Asp 195 200 205 Val Ala Phe Ile Arg Glu Ser Thr Val PheGlu Asp Leu Ser Asp Glu 210 215 220 Ala Glu Arg Asp Glu Tyr Glu Leu LeuCys Pro Asp Asn Thr Arg Lys 225 230 235 240 Pro Val Asp Lys Phe Lys AspCys His Leu Ala Arg Val Pro Ser His 245 250 255 Ala Val Val Ala Arg SerVal Asn Gly Lys Glu Asp Ala Ile Trp Asn 260 265 270 Leu Leu Arg Gln AlaGln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys 275 280 285 Phe Gln Leu PheGly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys 290 295 300 Asp Ser AlaIle Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly 305 310 315 320 LeuTyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys 325 330 335Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala 340 345350 Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser 355360 365 Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile370 375 380 Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp GlyGly 385 390 395 400 Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro ValLeu Ala Glu 405 410 415 Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp ProAsn Cys Val Asp 420 425 430 Arg Pro Val Glu Gly Tyr Leu Ala Val Ala ValVal Arg Arg Ser Asp 435 440 445 Thr Ser Leu Thr Trp Asn Ser Val Lys GlyLys Lys Ser Cys His Thr 450 455 460 Ala Val Asp Arg Thr Ala Gly Trp AsnIle Pro Met Gly Leu Leu Phe 465 470 475 480 Asn Gln Thr Gly Ser Cys LysPhe Asp Glu Tyr Phe Ser Gln Ser Cys 485 490 495 Ala Pro Gly Ser Asp ProArg Ser Asn Leu Cys Ala Leu Cys Ile Gly 500 505 510 Asp Glu Gln Gly GluAsn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr 515 520 525 Tyr Gly Tyr ThrGly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp 530 535 540 Val Ala PheVal Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn 545 550 555 560 AsnAsn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu 565 570 575Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys 580 585590 His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys 595600 605 Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe Gly610 615 620 Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln SerGlu 625 630 635 640 Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys LeuAla Arg Leu 645 650 655 His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly ProGln Tyr Val Ala 660 665 670 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr SerPro Leu Leu Glu Ala 675 680 685 Cys Glu Phe Leu Arg Lys 690 695 36 basepairs nucleic acid single linear protein NO NO 3 AGCGGATCCG GCCGTAGGAGAAGGAGTGTT CAGTGG 36 33 base pairs nucleic acid single linear DNA(genomic) NO NO 4 CGATCTAGAT TACTTCCTGA GGAATCCACA GGC 33 42 base pairsnucleic acid single linear DNA (genomic) NO NO 5 AAGCTTATGA AACTTGTCTTCCTCGTCCTG TTCTTCCTCG GG 42 35 base pairs nucleic acid single linearprotein NO NO 6 GATCCAGCCA GAGAGAGTCC GAGGGCCCCG AGGAA 35 28 base pairsnucleic acid single linear DNA (genomic) NO NO 7 ATTATTCGAA ACGAGGAATTAGCTTATG 28 22 base pairs nucleic acid single linear DNA (genomic) NO NO8 TCGAGAAAAG ACTTTTCTCC GG 22

1. A method for reducing the microbial contamination of a meat product,comprising treating the meat product with a sufficient amount oflactoferrin to reduce microbial contamination.
 2. A method for reducingthe microbial contamination of a meat product, comprising treating themeat product with a sufficient amount of isolated lactoferrin to reducemicrobial contamination.
 3. A method for reducing the microbialcontamination of a meat product, comprising treating the meat productwith a sufficient amount of lactoferrin mixed with a carrier to reducemicrobial contamination.
 4. A method for reducing the microbialcontamination of a meat product, comprising treating the meat productwith a sufficient amount of lactofernin mixed with a naturally occuringcarrier to reduce microbial contamination.
 5. A method for reducing themicrobial contamination of a meat product, comprising treating the meatproduct with a sufficient amount of isolated lactoferrin mixed with anaturally occuring carrier to reduce microbial contamination.
 6. Amethod in accordance with claim 1, wherein said lactoferrin is arecombinant lactoferrin.
 7. A method in accordance with claim 3, whereinsaid lactoferrin is a recombinant lactofernin.
 8. A method for reducingthe microbial contamination of a composition subject to microbialcontamination comprising treating the composition with a sufficientamount of lactoferrin to reduce microbial contamination.
 9. A method forreducing the microbial contamination of a composition subject tomicrobial contamination comprising treating the composition with asufficient amount of isolated lactoferrin to reduce microbialcontamination.
 10. A method for reducing the microbial contamination ofa composition subject to microbial contamination comprising treating thecomposition with a sufficient amount of isolated lactoferrin compoundedwith a carrier to reduce microbial contamination.
 11. A method forreducing the microbial contamination of a composition subject tomicrobial contamination comprising treating the composition with asufficient amount of isolated lactoferrin bound with a naturallyoccuring carrier to reduce microbial contamination.
 12. A method forretarding food-spoilage comprising applying to food an effective amountof lactoferrin having less than 25% metal loading.